Analysis of Metallo-?-lactamase Sequence Constraints at High Resolution ?-lactam antibiotics are the most often used antibacterial agents and an increasing incidence of resistance to these drugs is a critical public health concern. The most common mechanism of bacterial resistance is ?-lactamase-catalyzed hydrolysis of these drugs. The zinc metallo-?-lactamases (MBLs) catalyze the hydrolysis of a wide range of ?-lactam antibiotics including carbapenems and have emerged as an important source of resistance. The zinc-containing enzymes can be further subdivided into subclasses B1, B2 and B3 with subclass B1 ?-lactamases being the most widespread among bacteria. Within subgroup B1, the IMP-1, VIM-2 and NDM-1 enzymes are the most clinically relevant as sources of antibiotic resistance. In order to understand how mutations influence their function and evolution and to design inhibitors of MBLs, it is necessary to understand how the amino acid sequence determines the structure and function of these enzymes. This question will be addressed using a strategy of codon randomization and selection followed by deep sequencing. Individual codons in the IMP-1, VIM-2, NDM-1 MBLs as well as the CphA enzyme from subclass B2 will be randomized to create libraries containing all possible substitutions for the residue position. The importance of each position for enzyme structure and function will be determined by selecting functional clones from each library based on their ability to confer Escherichia coli resistance to a ?-lactam antibiotic of interest. Ultra-high throughput DNA sequencing of functional mutants will be used to provide high resolution information on the range of amino acid substitutions at each residue position that are consistent with resistance to the antibiotic used for selection and thereby determine the sequence requirements for enzyme function. The experiment will be performed for several ?-lactam antibiotics and the sequence requirements for each drug will be compared to identify residue positions where the wild type amino acid is required for hydrolysis of all ?-lactams as well as positions that exhibit altered sequence requirements depending on the antibiotic used for selection, i.e., residues that control substrate specificity. As these comparisons are made for each of the active site positions, a high resolution picture of the determinants of catalysis and substrate specificity for an MBL will emerge. This will allow an accurate estimate of the impact of any amino acid substitution at any active site residue on the substrate specificity and antibiotic resistance profile provided by an MBL. The information generated from deep sequencing will be extended by performing enzyme kinetics and X-ray crystallography for a number of enzymes with altered specificity to provide insights into not only which residue positions control specificity but also how they do so at the molecular level. The detailed knowledge of how active site residue positions contribute to ?-lactam hydrolysis will facilitate the design of inhibitors that interact with the critically importnt MBL residues.